life Article First mtDNA Sequences and Body Measurements for Rattus norvegicus from the Mediterranean Island of Cyprus Eleftherios Hadjisterkotis 1, George Konstantinou 2, Daria Sanna 3,*, Monica Pirastru 3 and Paolo Mereu 3 1 Agricultural Research Institute, P.O. Box 22016, Nicosia 1516, Cyprus; [email protected] 2 Society for the Protection of Natural Heritage and the Biodiversity of Cyprus, Keryneias 6, Geri 2200, Cyprus; [email protected] 3 Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/b, 07100 Sassari, Italy; [email protected] (M.P.); [email protected] (P.M.) * Correspondence: [email protected] Received: 12 June 2020; Accepted: 3 August 2020; Published: 5 August 2020 Abstract: Invasive species are the primary driver of island taxa extinctions and, among them, those belonging to the genus Rattus are considered as the most damaging. The presence of black rat (Rattus rattus) on Cyprus has long been established, while that of brown rat (Rattus norvegicus) is dubious. This study is the first to provide molecular and morphological data to document the occurrence of R. norvegicus in the island of Cyprus. A total of 223 black rats and 14 brown rats were collected. Each sample was first taxonomically attributed on the basis of body measurements and cranial observations. Four of the specimens identified as R. norvegicus and one identified as R. rattus were subjected to molecular characterization in order to corroborate species identification. The analyses of the mitochondrial control region were consistent with morphological data, supporting the taxonomic identification of the samples. At least two maternal molecular lineages for R. norvegicus were found in Cyprus. The small number of brown rats collected in the island, as well as the large number of samples of black rats retrieved in the past years might be an indication that the distribution of R. norvegicus is still limited into three out of the six districts of Cyprus. Keywords: mitochondrial DNA; D-loop; brown rat; alien species; biological invasion 1. Introduction Rattus rattus Linnaeus, 1758 (black rat) and Rattus norvegicus Berkenhaut, 1769 (brown rat) are considered as the most damaging and successful invasive species on the planet. Due to their commensality with humans they have spread in almost all regions of the world, with R. rattus occurring on all continents and R. norvegicus excluded only from Antarctica [1–3]. Invasive species, and more precisely species of the genus Rattus, are known to negatively affect island biota [4–6] and are the primary drivers of species extinctions on islands [7–9]. Due to the overall dramatic impact of rat invasions on autochthonous biota and on public health, the taxonomic identification of Rattus species spreading in specific locations, with particular reference to islands, are of pivotal importance for the conservation of local endemism [10,11]. The genus Rattus includes 66 species [3]; therefore, its taxonomy is complex being further complicated by a plethora of synonyms for different species [12]. Identification of individuals, even by experts, is often difficult [13], and for this reason the analysis of morphological traits combined with the genetic characterization of molecular markers is the most efficient system for a well addressed species taxonomic identification [3]. Life 2020, 10, 136; doi:10.3390/life10080136 www.mdpi.com/journal/life Life 2020, 10, 136 2 of 16 The island of Cyprus is situated at the eastern end of the Mediterranean Sea, south of Turkey, West of Syria, Lebanon and Israel, and North of Egypt, and it has been early and widely colonized by black rats since their spread from the Indian Peninsula [14,15]. On the contrary, the brown rat is not as frequent on the island and the earliest attempt to report species of Rattus for Cyprus was by Watson [16,17] who captured through trapping 150 rats from an area near Kyrenia. In addition, 1271 additional specimens were examined from various places along the north coastal region of the island. Watson concluded that “all the rats seen in Cyprus were the variety of brown back and white belly which is usually given the subspecies name of frugivorous” [17]. Nevertheless, Watson also stated that “the brown rat Rattus norvegicus also occasionally turns up in the ports but none were encountered elsewhere’. Similar outcomes were obtained by Spitzenberger [18] and Kourtellarides [19] who found R. rattus to be common in barn owl (Tyto alba) pellets in Cyprus, while no R. norvegicus were found in the island. In 1986–1987, one of the authors of the present study (E.H.) performed an extensive trapping of rats in Paphos Forest, an area of 620 km2, capturing only R. rattus (unpublished data). In 2003, in a list of the mammals of Cyprus, Hadjisterkotis [20,21] listed R. rattus and R. norvegicus as present on the island. However, the inclusion of R. norvegicus in this list was based strictly on oral information from Dr. A. Emanouel of the Central Veterinary Services of Nicosia, who noted that this species was present on the island only at the port of Limassol. Within the frame of a study on pathogens transmitted by rodents in Cyprus, rats were collected between 2000 and 2003 for the analysis of fleas [22,23]. Authors reported the wide trapping of hundreds of R. norvegicus and R. rattus, but although fleas were counted and identified using accepted morphologic criteria, no molecular or morphological data were provided for the collected rats. In 2009, Kryštufek and Vohralík [24] during their visit to Cyprus to study and record the small mammals of Cyprus, captured only black rats, in accordance with the findings reported by Watson [17]. Furthermore, these authors noted that R. rattus seems to be by far the most abundant small mammal in Cyprus, particularly so in the maquis and, to a lesser extent, in forests. Since they did not capture any R. norvegicus they noted that the presence of this species in Cyprus was dubious. Furthermore, Cucchi et al. [25] performed one of the most extensive live trappings for small mammals in Cyprus, noting the presence of only R. rattus, and concluded that this species was introduced with maritime traffic during historical times. From December 2006 to December 2009, the Research Promotion Foundation [26] of Cyprus, financed a project for the qualitative and quantitative study of wild small mammalian fauna in Natura 2000 sites of Cyprus, with the participation of the Wildlife Society of Cyprus, the University of Crete, and the Museum of Natural History of Crete, without finding any R. norvegicus on the island. In accordance, in 2016, Nicolaou et al. [27], after 9 years of live trapping of small mammals all over the island, including Limassol port, reported only the presence of R. rattus, and stated that “since the species lacks other rodent competitors in Cyprus, they also occur in areas remote from development”. A year later, Nicolaou [28] claimed that “in a number of projects referring to Cyprus, there are references for another rat species, R. norvegicus. However, there are no samples from this species until today to excuse its presence, although is possible that existed on the island in the past”. Accordingly, in the same year, Hadjisterkotis [29,30] found only R. rattus in the island, and in 2018, Moysi et al. [31] in a study on the diet of the barn owls on Cyprus, did not report any R. norvegicus, thus considering their presence on the island as dubious. During the last few years, several pictures of rats taken by bird watchers in Cyprus appeared on social media. The rats were usually identified as R. rattus, and occasionally the same images were reported as R. norvegicus. However, according to Kryštufek and Vohralík [24] and Nagorsen [32], while brown rat adults differ clearly from R. rattus, more slender juveniles with a pointed muzzle and relatively larger ears can be misidentified as black rats. Relative tail length, however, remains constant regardless of age [24]. Since the age of these rats is unknown, their identification as R. norvegicus based only on photographs, may be misleading. Although there are studies presenting body measurements of black rats from Cyprus [24], to the best of our knowledge, there are no molecular and/or morphological characterization of specimens belonging to the species R. norvegicus in Cyprus. Life 2020, 10, 136 3 of 16 In such a context, the primary aim of this study is the assessment of the presence of this species on the island, thus providing the first morphological characterization for individuals of R. norvegicus in Cyprus. In order to corroborate the morphological identification, a molecular taxonomic identification was also performed for several specimens, thus providing the first molecular data for this species in the island. 2. Materials and Methods 2.1. Sample Collection The number of rats collected and identified for each species is presented in Table1, and the sampling sites are indicated in Figure1. Rats were generally collected using snap traps and Herman live traps from January 2019 up to June 2020. Samples were also collected from animals found dead on the road. In addition, 18 skulls of R. rattus were collected from five barn owl nests, located at Xylophagou Larnaca, Machaeras Monastery, Kissonerga Paphos, Polis Paphos, and Vavatsinia Larnaca (Table1). Table 1. Sampling plan for the brown rats and black rats collected in the present study. + visual observation, ++ visual observation, dead in the nest of a long-legged Buzzard (Buteo rufinus), * road killed, ** captured in snap traps